Substrate composition and method for solid phase urease immunoassay

ABSTRACT

A method for enzyme immunoassay of a ligand includes urease as a label. A bound fraction of ligand and antiligand conjugated to urease is formed on a solid support. The urease component of the bound fraction is contacted with a substrate composition for urease which includes a compound converted to ammonia by the urease, a tetrazolium salt and a pH dependent reducing agent which reduces the tetrazolium salt when the pH of the assay medium has been raised by the ammonia. The tetrazolium salt is reduced to a colored insoluble formazan which precipitates as a detectable spot on the support. The invention includes the substrate composition and a kit of materials for performing the assay.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to immunoassay for a ligand, and moreparticularly relates to membrane immunoassay and particular reagentsuseful therein.

2. Background of the Invention

Assay systems which are both rapid and sensitive have been developed todetermine the concentration of a substance, generally referred to as theanalyte, present in low concentration in a fluid sample. Immunoassaysdepend on the binding of an antigen or hapten to a specific antibody andhave been particularly useful because they give high levels ofspecificity and sensitivity. These assays employ one of the abovereagents in labeled form, the labeled reagent being referred to as thetracer.

Enzymes have often been used as labels in immunoassay. In conventionalenzyme immunoassay (EIA), an enzyme is covalently conjugated with onecomponent of a specifically binding antigen antibody pair, and theresulting enzyme conjugate is reacted with a substrate to produce asignal which is detected and measured. The signal may be a color change,detected with the naked eye or by a spectrophotometric technique, or maybe conversion of the substrate to a product detected by fluorescence.

A convenient format for EIA is solid phase immunoassay in which one ofthe assay reagents is immobilized on a solid support. The solid supportmay be in the form of a dipstick, the inside wall of a test tube orcuvette or the well of a microtiter plate. A particularly useful solidsupport is a microporous membrane.

Membrane immunoassay is often referred to as flow through assay.Examples of flow through EIA wherein flow is generated by capillaryaction are the assays described in U.S. Pat. No. 3,888,629 to Bagshaw,U.S Pat. No. 4,246,339 to Cole et al. and U.S. Patent No. 4,632,901 toValkirs et al. U.S. Pat. No. 4,277,560 to Gray and U.S. Pat. No.4,812,293 to McLaurin et al. are examples of flow through assays usingpressure and vacuum respectively.

In membrane EIA, any number of liquids may be caused to flow through themembrane to effect binding, separation and washing of assay components.The final step in most membrane EIA procedures is contacting a colordeveloping reagent, such as a chromogen, with the membrane. Thechromogen reacts with enzyme captured on the membrane to produce acolored product which may be detected as evidence of the presence ofanalyte or measured as evidence of the concentration of analyte. Thecolored product may be soluble, in which case it will pass through themembrane and be detected in the filtrate, or it may be insoluble andform a colored spot on the membrane.

The enzyme urease converts urea into carbon dioxide and ammonia. It hasbeen developed as a label for solution immunoassay wherein a rise in pHof the assay medium due to the ammonia production is detectedcolorimetrically with an indicator such as bromcresol purple (Chandleret al., Journal of Immunological Methods 53, 187 (1982); U.S. Pat. No.4,590,157).

EIA in which urease is detected colorimetrically in solution by theChandler et al. procedure provides an excellent visual readout becausethe detected product is deeply colored and water soluble. On the otherhand, rapid diffusion due to the water solubility precludes depositionof the product as a spot on a solid phase, such as a dipstick or amembrane. This severely limits usefulness of urease in solid phase EIAprocedures. There is a need for a urease substrate which would beconverted to an insoluble product to be deposited on a solid phase. Sucha substrate would greatly extend the usefulness of urease as animmunological label. The present invention provides such a substrate.

SUMMARY OF THE INVENTION

One aspect of the invention is a method for enzyme immunoassay of aligand. A liquid sample suspected of containing the ligand is incubatedwith a solid support having affixed thereto a capture antiligand wherebythe ligand is bound to the support. The support may then be contactedwith a tracer comprising a detection antiligand conjugated to urease sothat urease becomes affixed to the support. A substrate composition forthe urease is then brought into contact with support. Urease converts afirst component of the composition to ammonia. The ammonia raises the pHof the assay medium sufficiently to activate a second component of thecomposition, a pH dependent reducing agent. When activated, the reducingagent reduces a third component, a tetrazolium salt, to a coloredinsoluble formazan which precipitates as a detectable spot on thesupport.

A preferred support is a porous membrane which preferably is mounted ina suitable holder adjacent an absorbent pad. The pad causes liquids toflow through the membrane by capillary action.

The preferred ligand for assay is an antigen, most preferably a viralantigen, and the preferred capture antiligand is an antibody. Thepreferred tracer is a second antibody, referred to as the detectionantibody, conjugated to urease. The preferred substrate compositionincludes urea as the ammonia generating compound and ascorbic acid asthe reducing agent which reduces the tetrazolium salt when the liberatedammonia causes a rise in the pH.

Thus, the assay system of the invention provides significant improvementin immunoassay using urease as the label. First and foremost, thesubstrate composition provides a colored detectable product whichprecipitates on a solid support in contrast to all urease assays in theprior art which are solution assays. The color formed is stable and notreversible, so that the enzymatic reaction can be stopped with a washsolution without loss or change in the color. In contrast, urease assaysof the prior art using pH indicators such as bromcresol purple or phenolred give reversible color changes. The invention takes advantage of thefast turnover rate of urease and overcomes the limitation in the priorart of solution assay, making possible commercial development of aurease assay by the far easier and more dependable solid phase format.

BRIEF DESCRIPTION OF THE DRAWING

The figure illustrates detection of urease activity by the method of theinvention as a function of incubation time.

DETAILED DESCRIPTION

While this invention is satisfied by embodiments in many differentforms, there will herein be described in detail preferred embodiments ofthe invention, with the understanding that the present disclosure is tobe considered as exemplary of the principles of the invention and is notintended to limit the invention to the embodiments described. The scopeof the invention will be measured by the appended claims and theirequivalents.

One aspect of the present invention is a substrate composition which isconverted by the urease to a colored insoluble product. A second aspectof the invention is a method for immunoassay of a ligand using urease asthe label and the composition as substrate. The method takes advantageof the many attributes of urease as a label and at the same timeovercomes the drawback which has heretofore prevented this enzyme fromachieving widespread use in immunoassay.

The assay of the invention may be performed by any conventional solidphase technique in which the presence or absence of a ligand in a sampleis detected by enzyme catalyzed conversion of a substrate to a coloredproduct. For example, the assay of the invention may be performed byimmunochromatography. In this procedure, a liquid phase containingligand to be detected migrates by capillary action across a solid phase,such as a glassplate, having various assay components deposited onadjacent but separated zones thereof. Representative of this procedureare the assays disclosed in U.S. Pat. No. 4,740,468 to Weng et al. andU.S. Pat. No. 4,446,232 to Liotta.

Another suitable assay technique uses a dipstick. In this procedure, asolid phase, usually a glass plate having a binder containing a captureantibody thereon, is dipped alternately into the test liquid, liquidscontaining assay reagents and wash liquids. In the last dip, an enzymecaptured on the dipstick in proportion to the concentration of ligand inthe test liquid converts the substrate of the invention to a coloredproduct which deposits on the dipstick and which is indicative of thepresence of ligand.

While immunoassay for a ligand as described above is a preferredapplication of the invention, one skilled in the art will immediatelyrecognize that the method may be used in an assay wherein the ligand maybe a nucleic acid probe and the tracer may be a complementary strand ofDNA or RNA conjugated to urease. Assay procedures having enzymesconjugated to DNA and RNA strands are well known in the art.

A preferred assay technique is flow through assay in which the solidphase is a porous membrane. The membrane may be positioned in anysuitable assay device adapted for flow through assay as known in theart. In preferred devices, flow of assay liquids is promoted bycapillary action induced by a pad of absorbent material adjacent themembrane, and the membrane and absorbent pad are mounted in a suitablehousing. Membrane flow-through assay and various devices therefor havebeen disclosed and several devices are commercially available.

The porous membrane may be of any material which does not interfere inany way with any other component or step of the assay. Suitablemembranes are, for example, of glass fiber, polyvinylidene difluoride,polycarbonate, nitrocellulose and nylon. Such membranes are well knownin the art and many are commercially available from suppliers such asPall (East Hills, New York), Millipore (Bedford, Massachusetts) andSchleicher and Schuell (Keene, New Hampshire).

The ligand may be from any source, and may be an antigen, an antibody ora hapten. For example, the ligand may be an endocrine hormone, such asHCG or FSH, present in body fluid, or it may be isolated from a bodyfluid and subsequently introduced into a different liquid, such asbuffer. In other cases, the ligand may be from a source other than abody fluid, as, for example, a culture of microorganisms such asChlamydia or a cellular extract thereof. Antibodies, such as theantibody against Lyme disease, may be assayed, or the ligand may be ahapten such as a therapeutic drug or a drug of abuse.

Preferred ligands are antigens, most preferably viral antigens presentin a body fluid, such as Adenovirus, Parainfluenza 3 virus and, mostpreferably, Herpes simplex virus (HSV), Respiratory syncytial virus(RSV), and Influenza A (Flu A). The invention will hereinafter bedescribed generically in terms of the preferred membrane assay.

The membrane may be coated with an antiligand specific for the ligand.Thus, in the case where the ligand is the preferred viral antigen, theantiligand may be an antibody which binds specifically to the antigenand thereby captures the antigen on the membrane. This reagent ishereinafter referred to as the capture antibody. The membrane may befurther coated with an inert protein to fill any binding sites on themembrane not occupied by the capture antibody. (In the presentdisclosure, the term inert protein means a protein which isimmunologically unreactive toward any other component of the assay andwhich does not substantially bind nonspecifically to other proteins inthe assay medium, with the understanding that the inert protein may wellbe immunologically reactive toward other materials which are not part ofthe assay of the invention.) Representative nonlimiting examples ofsuitable inert proteins are casein and albumin, although others will beevident to those skilled in the art.

If the ligand is a hapten, it may be necessary to conjugate the haptento a protein in order to raise a suitable anti-hapten capture antibody.Such procedures are well known in the art of hapten immunoassay, andfurther details with respect to this aspect of the invention are notneeded for a complete understanding of the invention.

Coating of the membrane with either or both of the capture antibody orthe inert protein may be carried out by any suitable method, preferablyby incubating the membrane with a solution of the antibody and/or inertprotein whereby the protein is physically absorbed into the polymericmatrix of the surface of the membrane. Coating procedures are whollyconventional in the art.

The membrane may be incubated with the sample suspected of containingthe ligand in order to bind the ligand to the antiligand coated onto themembrane. Preferably the sample is applied to the coated membrane andallowed to pass through the membrane in a transient, flow through formatfor about 1 to 15, preferably about 5 minutes at a temperature of about0° to 50° C., preferably about ambient temperature. By this procedure,antigen in the sample is captured on the membrane in proportion to itsconcentration in the sample. In addition, it has been found that viralantigen is absorbed preferentially even when the sample contains a largeexcess of extraneous protein, such as is the case when the sample is abody fluid.

In an alternate embodiment of the invention, the membrane may be coatedwith the inert protein and the antigen absorbed directly onto thissurface and the assay performed without a capture antibody. Flow throughimmunoassay absent a capture antibody is disclosed in copendingapplication serial number 272,380, filed Nov. 17, 1988, of commonassignee herewith. In still another embodiment, the antigen may beadsorbed directly onto the membrane, and the membrane containing antigensubsequently treated with the inert protein to fill all unoccupiedbinding sites.

The membrane having ligand bound thereto may be treated with a solutionof the tracer. The tracer may be an antiligand, hereinafter referred toas the detection antiligand, conjugated to urease wherein the assay isperformed by the conventional sandwich or half sandwich technique. Thepreferred detection antiligand is an antibody which binds to antigencaptured on the membrane and thereby affixes urease to the membranesurface in direct proportion to the quantity of antigen in the sample.Alternatively, the urease may be conjugated by conventional methods to abinder such as biotin, avidin and streptavidin and the latter bound tothe antiligand.

The assay may also be performed by competitive assay, in which case thetracer may be the ligand conjugated to urease or to a conjugate ofurease with biotin, avidin or streptavidin. In this format, the ligandand tracer compete for antiligand binding sites, and the urease becomesaffixed to the membrane surface in inverse proportion to the quantity ofligand in the sample. Competitive assay may also be carried out with atracer comprising urease linked to a hapten which has been used to raisethe detection antibody, preferably a monoclonal antibody.

Conjugation of enzymes, such as urease, or enzyme conjugates withbiotin, avidin or streptavidin to antigens or antibodies is well knownin the art and deemed to be within the purview of one skilled in the artwith no further detail.

The membrane having urease affixed thereto may be treated with thesubstrate composition of the invention. The composition may include atleast three components which interact consecutively leading to a coloredinsoluble product. The first component of the composition is a compoundconverted by the urease to a pH-raising substance. Suitable compounds asknown in the art are urea, substituted ureas such as, for example, Nmethylurea and semicarbazide, and simple amides such as formamide andacetamide. Urease cleaves these compounds to ammonia which raises the pHof the assay medium. The preferred first component is urea.

The second component of the substrate composition may be a pH dependentreducing agent for the third component. Suitable second components areindoxyl and, preferably, ascorbic acid. The second component does notreduce the third component, a tetrazolium salt, at low pH. Liberation ofammonia by the action of urease on the first component, however, raisesthe pH sufficiently to trigger the reduction of the tetrazolium salt toa colored, insoluble formazan. Reduction of tetrazolium salts toformazans is well-known in the art. Representative suitable tetrazoliumsalts are iodonitrotetrazolium violet and nitrobluetetrazolium chloride,although others are well-known to those skilled in the art.

Thus, in the preferred sandwich assay of the invention, ligand in thesample is captured by the antiligand coated onto the membrane. Capturedligand binds to the tracer whereby urease becomes affixed to themembrane. Urease affixed to the membrane converts the urea in thesubstrate composition to ammonia. The ammonia raises the pH to a levelat which the ascorbic acid reduces the tetrazolium salt to the formazan.Accordingly, in the sandwich assay format, the appearance of color onthe membrane is indicative of ligand in the sample.

In a competitive assay, ligand in the sample competes with tracer forbinding to the antiligand. Thus, in this assay configuration, tracer,and therefore, urease, becomes affixed to the membrane in inverseproportion to the concentration of ligand. Accordingly, in a competitiveassay, absence of colored formazan is indicative of ligand in thesample.

It is evident that the method of the invention may be used to assay forurea. For this embodiment of the invention, urease may be adsorbed onthe membrane and a solution containing an unknown amount of urea,ascorbate and a tetrazolium salt may then be passed through themembrane. Urea in the unknown causes a spot to appear on the membrane.The color may be compared to standards containing known quantities ofurea to quantitate the urea.

The following examples are provided to further describe the inventionbut are in no way to be considered as limitative of the invention.

EXAMPLE I Detection of Urease Using SodiumAscorbate/Iodonitrotetrazolium Violet (Biodyne C Membrane)

A membrane filter stack was assembled with the following configuration:

Top Layer -- Three micron Biodyne® C Membrane, (Pall, Glen Cove, NewYork, #BIA0030HC5). Precoated by immersion in phosphate buffered salinecontaining 0.3% casein for 30 minutes at ambient temperature.

Next Layer -- Non woven rayon sheet (Schleicher and Schuell, Keene, NewHampshire; #5-S)

Bottom Layer -- Cellulose absorbent pads (2) (Filtration Sciences, MountHolly Springs, Pennsylvania; #ED 320-200)

The membrane layers were encased in a plastic holder which includes areceiving well formed above the top layer. Within this well was fitted aflow restriction insert which has an aperture more narrow than thereceiving well and sits flush against the top membrane.

A solution of urease biotin conjugate in TEOA buffer (150 μl, 0.1 Mtriethanolamine, 1 mM EDTA, pH 7.6) was added through the restrictioninsert to produce a triangular pattern of adsorbed urease biotinconjugate on the membrane. After one minute, the insert was removed andthe membrane washed with TEOA buffer (200 μl). An aqueous solution ofiodonitrotetrazolium violet (200 μl, 0.2 mg/ml) was added, followed byurease substrate (200 μl, 25 mM urea, 1 mM EDTA, 20 mM sodium ascorbate,pH 5.0). After 10, 20 and 30 minutes, stop buffer (400 μl, 150 mM sodiumcitrate, pH 3.0) was added and the membrane color determined using aGretag reflectometer (magenta setting). This color was observed as atriangular pattern corresponding to adsorbed urease biotin conjugate.Samples without added urease showed no triangular pattern on themembrane.

The relationship between the time of incubation and the limit of ureasedetection is given in the Figure.

EXAMPLE II Detection of Urease Using SodiumAscorbate/Nitrobluetetrazolium Chloride (Biodyne C Membrane)

The procedure in Example I was followed except for the substitution ofaqueous iodonitrotetrazolium violet by nitrobluetetrazolium chloride(300 μl, 0.165 mg/ml, 0.5% methanol). After twelve minutes, the membranecolor was determined used a Gretag reflectometer (black setting). Thedetection limit for urease-biotin was 10 ng/test. Membranes withoutadded urease showed no triangular pattern.

EXAMPLE III Detection of Urease Using Indoxyl/NitrobluetetrazoliumChloride (Biodyne C Membrane)

Urease biotin conjugate was adsorbed onto Biodyne C membranes asdescribed in Example I. The insert was removed and the sample washedwith TEOA buffer (200 μl). Nitrobluetetrazolium chloride was added as inExample II. Urease substrate solution (25 mM urea, mM, EDTA, 1 mMindoxyl butyrate) was treated with rabbit liver esterase (5.3 μg/ml) togenerate indoxyl from indoxylbutyrate. The solution was mixed brieflyand added to the device. After twelve minutes, stop buffer was added andthe membrane color was read using a Gretag reflectometer (blacksetting). The detection limit for urease biotin was 10 ng/test. Colorwas observed only where the urease biotin conjugate was bound to themembrane.

EXAMPLE IV Assay for Urea Using Tetrazolium Salt Reduction

Devices are prepared as described in Example I. A solution of urease inTEOA buffer is adsorbed onto the Biodyne C membrane. An aqueous solutionof iodonitrotetrazolium violet (200 uL, 0.2 mg/mL) is added and allowedto flow through the device. A sample containing an unknown quantity ofurea is passed through the membrane and standards having knownconcentrations of urea in substrate buffer (1 mM EDTA, 20 mM sodiumascorbate) are applied to other membranes having the same amount ofadsorbed urease. After five minutes the intensity of color in theunknown is measured with a reflectometer and correlated to the amount ofurea present in the standards. This correlation is in turn used todetermine the concentration of urea present in unknown samples assayedby the above method.

What is claimed is:
 1. A method for detecting a ligand in a liquidcomprising:a) passing a liquid suspected of containing said ligandthrough a porous membrane precoated with a capture antiligand such thatsaid ligand is captured on said membrane; b) passing through saidmembrane a tracer solution comprising urease conjugated to a moleculeselected from the group consisting of the ligand and a detectionantiligand such that said urease becomes affixed to said membrane; andc) passing through said membrane a solution comprising (i) a compoundconverted by said urease to ammonia, (ii) a pH-dependent reducing agentselected from the group consisting of ascorbic acid and indoxyl and(iii) a tetrazolium salt, said urease affixed to the membrane convertingsaid compound to ammonia, said ammonia raising the pH of the solutionthereby activating said reducing agent, the activated reducing agentreducing said tetrazolium salt to a formazan, said formazanprecipitating as a colored spot on said membrane and indicating thepresence of the ligand in said liquid.
 2. The method of claim 1 whereinsaid ligand is selected from the group consisting of an antigen, anantibody and a hapten.
 3. The method of claim 2 wherein said ligand isan antigen or a hapten and said capture antiligand is an antibodyspecific for said antigen or said hapten.
 4. The method of claim 2wherein said ligand is an antibody and said capture antiligand is anantigen specific for said antibody.
 5. The method of claim 1 whereinsaid tracer comprises urease conjugated to the ligand, and said ligandand said tracer compete for binding sites on said capture antiligand. 6.The method of claim 1 wherein said tracer comprises urease conjugated tosaid detection antiligand and said tracer to said ligand.
 7. The methodof claim 1 wherein said compound is selected from the group consistingof urea, N-methyl urea, semicarbazide, formamide and acetamide.
 8. Themethod of claim 1 wherein the precoated membrane is prepared by coatingwith said capture antiligand and coating with an inert protein to blockunfilled binding sites on the precoated membrane.
 9. The method of claim8 wherein said inert protein is selected from the group consisting ofalbumin and casein.
 10. The method of claim 1 wherein said tetrazoliumsalt is selected from the group consisting of iodonitrotetrazoliumviolet and nitrobluetetrazolium chloride.
 11. A method for detecting aligand in a liquid comprising:a) contacting a liquid suspected ofcontaining said ligand with a solid support such that said ligandattaches to said support; b) contacting the ligand attached to thesupport with a tracer comprising urease conjugated to an antiligand suchthat said antiligand binds to the attached ligand and said ureasebecomes affixed to said support; and c) contacting the urease affixed tosaid support with a substrate composition for said urease, saidcomposition comprising a compound converted by said urease to ammonia, atetrazolium salt and a pH-dependent reducing agent for said tetrazoliumsalt selected from the group consisting of ascorbic acid and indoxylwhereby production of ammonia by said urease causes a rise in pHsufficient to activate the reducing agent such that the tetrazolium saltis reduced by said activated reducing agent to a colored formazan whichforms a colored spot on said support of said ligand is present in saidliquid.
 12. The method of claim 11 wherein said support is a dipstick.13. The method of claim 11 wherein said support is a porous membrane.14. The method of claim 11 wherein said support is animmunochromatography plate.
 15. A method for detecting an antigen in aliquid comprising:a) combining a liquid suspected of containing theantigen with a porous membrane coated with a capture antibody and with adetection antibody conjugated to urease such that said antigen binds tosaid capture and detection antibodies to give a membrane bound fractioncomprising said urease on said membrane; b) allowing said liquid to passthrough said membrane; and c) passing a substrate composition comprisingurea, ascorbic acid and a tetrazolium salt through said membrane, saidurease bound on the membrane converting said urea to ammonia, saidammonia activating said ascorbic acid to reduce said tetrazolium salt toa formazan, said formazan precipitating as a colored spot on saidmembrane and indicating the presence of said ligand in the liquid. 16.The method of claim 15 wherein the coated membrane is prepared bycoating with said capture antibody and coating with an inert protein toblock unfilled binding sites on the coated membrane.